DNA methylation profiling of human chromosomes 6, 20 and 22

This paper was found while I have been searching Epignome Project. This is the last paper which was published from Epigenome Project, and I decided to present this on next journal club meeting. This paper have been cited by 317 articles.


The really interesting discovery from introduction of this paper, Epigenome project was dubbed to AHEAD (the Alliance for Human Epigenomics and Disease). And there are some clue (http://www.nature.com/nature/journal/v454/n7205/full/454711a.html, http://www.aacr.org/home/scientists/working-groups--task-forces/task-forces/human-epigenome-task-force.aspx). I will summarize this on next time.


Result

In this paper, they report the methylation profiling of human chromosomes 6, 20 and 22 in 43 samples derived from 12 different normal tissues. They controlled age and sex, which can affect the methylation state, in each samples. 2,524 amplicons with 1.88 M CpG sites on chromosome 6, 20 and 22 that are associated with 873 genes were sequenced by sanger sequencer.

Distribution of methylation
bimodal distribution of amplicon's methylation state, 27.4% unmethylated(<20% methylation), 42.2 hyper-methylated(>80% methylation) and 30.2 heterogeneously methylated(20%~80%). Only 9.2 of CGIs were hyper-methylated.

By distinction  mosaicism versus imprinting among heterogeneously methylated amplicons, they confirmed the most of them were by mosaicism.

Comethylation (relationship between the degree of methylation over distance) was quite strong over short distances(<1000 bp). This fact showed that in normal the range of comethylation was shorter than in specific diseases. Absolute difference in methylation betweens tissues were shown in figure. Sperm stood out, and related tissue showed low level of difference.




Promoter methylation

They divide promoter region in three types. 5' UTR, putative TSSs, Sp1(transcription factor) binding site. And 5' UTR are sub-divided according existence of CGIs, and gene type (novel CDS, novel transcript, pseudo gene).
The most CGIs in 5' UTR are unmethylated, while non CGI 5' UTRs are hyper-methylated. There is no big difference between CDS, transcript, pseudo in exonic region, but in 5' UTR the most amplicon which belong to CDS are unmethylated.

TSSs showed unmethylated core region of about 1,000 bp.

Age- and sex-dependent DNA methylation


They compared methylation state according to age (26+-4 age, 68+-8 age) and sex, but couldn't find any significant difference.


Differential methylation


T-DMRs (tissue-specific differentially methylated regions) was confirmed by hierarchical clustering which showed biological replicate of each tissue grouped together. 22 % of amplicons were T-DMRs. The frequency of T-DMRs which belong to CGIs was low.

They found that T-DMRs in 5' UTRs which are without CGIs could affect mRNA expression. T-DMRs located in gene have little effect on mRNA expression.


Conservation of DNA methylation

To check conservation of methylation across species, they compare 59 orthologous amplicons between human and mouse. The majority of profiles were conserved. They extrapolate that 70  % of orthologous loci between human and mouse may have conserved methylation profiles.








Like most papers in early stage of high-resolution sequencing for methylation state, This paper also just showed landscape of methylation state over large region in chromosomes. There were just arbitrary classification of methylation state and correlation this classification with some specific conditions. There is no absolute rule.




I think that It will be fun that finding orthologous genes which show different methylation state between human and mouse. Are these genes species-specific genes?